A confocal scanning microscope can be used, for example, to excite fluorescing substances in samples, in particular tissue samples, and to detect the fluorescent light resulting therefrom. The principle is that an illumination light beam is directed via a scanning unit onto a sample, the scanning unit being operated with a driving signal that encompasses scan coordinate values. The scan coordinate values are representative of positions of positioning elements of the scanning unit. The fluorescent light radiated from the sample is directed, via the scanning unit and a beam splitter that separates the fluorescent light from the illumination light, onto a detection pinhole that allows a small portion of the detected light to pass through to a detector. The detected light sensed with the aid of the detector is detected as a function of the scan coordinate values. In other words, in addition to the signal detected by the scanning unit, the position occupied by the positioning elements of the scanning unit upon detection of the detected light is also recorded, the positions being represented by the scan coordinate values.
The detected light can be detected as a function of the scan coordinate values with which the scanning unit is driven; if the scanning unit (in particular its positions) is controlled in closed-loop fashion, the predefined scan coordinate values can also be referred to as “target” scan coordinate values. Alternatively thereto, true positions of the scanning unit can be sensed with the aid of sensors, and these sensed scan coordinate values can then be associated with the detected light detected at the sensed positions; once again, if the positions of the scanning unit are controlled in closed-loop fashion, these sensed scan coordinate values can also be referred to as “actual” scan coordinate values.
During optical scanning of the sample or thereafter, the image of the sample can be assembled on the basis of many acquired image points, the position of each image point being determined by a pair of image coordinate values, and corresponding scan coordinate values being associated with the image coordinate values. It is known to determine this association of the image coordinate values with the scan coordinate values by means of approximations. This can result, however, in distortions and/or deformations, in particular at the edges of the image of the sample.
The distortions of the images occur not only because of the approximately calculated scan coordinate values, but also because of optical effects of the optical elements of the confocal scanning microscope which are not considered in known scanning microscopes or methods for operating the scanning microscopes.
The distortions are problematic in particular when an overall image of a sample is to be assembled from multiple juxtaposed individual images, since the images then do not match one another at their edges. This can furthermore be problematic when the image of the sample, acquired confocally in this fashion, is to be overlaid on a wide field image, since such distortions at the image edges do not occur with wide field imaging and the two images of the sample thus do not fit together, especially at the edges of the images.